INFECTION AND IMMUNITY, May 1972, p. 814-825 Vol. 5, No. 5Copyright (©) 1972 American Society for Miicrobiology Pri,ited in U.S.A.

Activity of Macrophage and Neutrophil CellularFractions from Normal and Immune Sheep Against

Listeria monocytogenes

P. M. OUTTERIDGE,l J. W. OSEBOLD, AND Y. C. ZEEDepartmelnt of Veteriniary Microbiology, Sc/hool of Veteriniary Medicinie, Uniiversity of California,

Davis, Californiia 95616

Received for publication 21 October 1971

Cellular immunity to Listeria monocytogenes infection was studied by assaying forantibacterial activity in fractions of leukocytes collected from the peritoneal cavity,lungs, and mammary glands of immunized sheep. The cells were collected in popu-lations that were largely either macrophages or neutrophils. Mechanically disruptedcells were divided into nuclear, lysosomal, and supernatant fluid fractionsand then subjected to freezing and thawing. Comparison with similarly treatedrabbit cells showed that greater fragility exists in the lysosomes of sheep cells, asindicated by the amount of acid phosphatase activity released. Inhibition of bac-terial growth was assayed in a broth medium at pH 4.6. As expected, nuclear andlysosomal fractions from neutrophils were inhibitory. Some antibacterial activitywas found in nuclear fractions of macrophages. The lysosomes of macrophagescollected from the peritoneal cavity and the mammary gland did not inhibit thegrowth of L. monocytogenes. Peritoneal macrophages were allowed to interactwith sensitized lymphocytes and an avirulent strain of L. monocytogenes for 4 hrprior to disruption and fractionation, but antibacterial activity was not detected.Pulmonary alveolar macrophages from 5 out of 16 sheep contained Listeria inhibi-tory activity in their lysosomes. The mechanism was inhibitory but not bactericidal.

The term "cellular immunity" may be used todescribe the increased capacity of macrophages tokill or to inhibit the growth of intracellular micro-organisms. Diseases in which this occurs includethose caused by facultative intracellular bacteriasuch as Mycobacterium, Brucella, Listeria, andSalmonella species (8, 17, 21, 32).The mechanism of cellular immunity remains

undefined, but it has been attributed to a non-specific hyperactivation of macrophages in lesionsites (6), to cytophilic antibodies on macrophages(13, 31), and to delayed hypersensitivity (19). Invitro studies on the interaction of macrophagesand Listeria monocytogenes have shown that the"immune cells" resist the destructive effects of theorganism and also suppress bacterial growth(18, 21). More recent investigations have indi-cated that mice can be stimulated by infectionwith Listeria to exhibit cross-resistance to Myco-bacterium, suggesting that cellular immunity may

I Present address: Commonwealth Scientific and IndustrialResearch Organization, Division of Animal Health, Parkville,Victoria 3052, Australia.

not be immunologically specific (5). liowever, thesame study also showed that mice had greaterresistance against the organism to which they hadbeen immunized. Youmans (36) has describedevidence to show that macrophages from im-munized animals possess both a nonspecific re-sistance and a specifically directed immune cellmechanism for intracellular killing.

It has been noted that macrophages from bothnormal and immune animals have increasednumbers of lysosomes in their cytoplasm duringin vitro incubation (3, 10) and following infec-tion with Listeria monocy togenes (23). Theneutrophil granule has been shown to fuse withphagocytic vacuoles (40), and this is followed bythe killing of bacteria inside the vacuole (2). Ithas been suggested by Zeya and Spitznagel (37)that the intracellular killing by neutrophils is dueto the release of bactericidal cationic proteinsfrom granules into the phagocytic vacuole. North(22) showed that mouse macrophage lysosomesfuse with phagocytic vacuoles containing Listeriawith the release of lysosomal contents.

814

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PHAGOCYTE FRACTIONS AGAINST LISTERIA

However, there is a notable lack of reports onthe bactericidal properties of macrophage gran-ules. It seemed important to investigate the inter-action of these granules with bacteria and, inparticular, the granules from those cells exhibitingthe increased resistance associated with cellularimmunity. Studies are reported here using macro-phages from normal sheep andListeria-immunizedsheep. Sheep were chosen as the experimentalanimal since they are commonly subject to naturalListeria infections and cellular immunity hasbeen demonstrated in vitro with the peritonealexudate cells of immunized sheep (21). Macro-phages derived from the peritoneal cavity, lungs,and mammary glands were investigated.

Cellular fractions from macrophages were com-pared with fractions obtained from the neutro-phils of sheep and rabbits.

MATERIALS AND METHODS

Animals. The sheep were of cross-bred Hampshire-Rambouillet stock. Animals were obtained from prem-ises where listeriosis had never been encountered, andthey were serologically tested to demonstrate an ab-sence of mercaptoethanol-resistant agglutinatingantibodies to L. monocytogenies (24). Leukocytes wereobtained from the mammary glands of virgin ewesthat were free of mastitis. New Zealand White rabbitswere also used as a source of leukocytes.

Bacteria. A virulent strain of L. monocytogenies(strain 3-54, serotype 4b) was used in antibacterialassays and for the immunization of sheep. The strainwas originally isolated from the brain of a naturallyinfected sheep and was frequently passaged throughmice to maintain its virulence.

Immunization of sheep with Listeria. Immunemacrophage production was stimulated in sheep bysubcutaneous and intraperitoneal inoculations of liv-ing L. moniocytogenes. The bacteria were grown for18 hr in Trypticase soy broth (Baltimore BiologicalLaboratory), centrifuged at 15,000 X g for 25 min,washed once in Zobell's solution (39) (pH 7.0), andthen resuspended in Zobell's solution. Numbers ofbacteria were estimated photometrically, and bacteriawere inoculated in three or more doses ranging from2 X 10' to 4 X 10' per kg of body weight at approxi-mately 7-day intervals (actual doses were 1.1 X 1010to 2.7 X 1010).

Leukocytes. All leukocytes were harvested in sterile0.25 M sucrose solution to which sodium heparin hadbeen added at a concentration of five units per ml.

Cells were counted on a hemocytometer. Differen-tial cell counts were made on Giemsa-stained prepara-tions.

Neutrophils. Neutrophils were obtained from thenonlactating mammary glands of ewes after the in-fusion of 5,ug of bacterial lipopolysaccharide (Es-chlerichia coli 055:B5, Difco) into each gland via thestreak canal (26). The migration of leukocytes intothe glands was thus stimulated, and the cells werewashed out after 6 hr with 10 ml of sterile 0.25 M

sucrose solution and collected in sterile centrifugetubes.

Neutrophils were also obtained from the peritonealcavities of rabbits 6 hr after the infusion of shellfishglycogen (Mann Research Laboratories). The glyco-gen was sterilized with ethylene oxide and preparedin a concentration of 1 mg per ml in sterile 0.25 Msucrose solution. A 300-ml amount of this solutionwas injected intraperitoneally per rabbit. The cell-laden fluid was obtained by paracentesis from therabbits under ether anesthesia.

Macrophages. Macrophages from the mammaryglands were collected from the same ewes that pro-vided neutrophils by again washing out the glandswith 10 ml of sterile 0.25 M sucrose solution 5 daysafter the infusion of bacterial lipopolysaccharide (26).

Peritoneal macrophages were collected from theewes 5 days after injecting 200 ml of sterile mineraloil (Drakeol 35VR, Pennsylvania Refining Co.) intra-peritoneally. The peritoneal exudate cells were washedout in cold 0.25 M sucrose solution after slaughter ofthe animal.Lung alveolar macrophages were obtained after

removing the lungs from sheep which had been ex-sanguinated. The lungs were suspended by a clampattached to the trachea, and sterile 0.25 M sucrosesolution was infused into the bronchi. Endobronchiallavage was repeated several times, and the cell-ladenliquid was collected by pouring the lung fluid into asterile flask.

Peritoneal macrophages were obtained from rabbits5 days after intraperitoneal injection of 50 ml ofsterile mineral oil. The cells were obtained fromanesthetized rabbits by introducing 300 ml of sterile0.25 M sucrose solution into the peritoneal cavity. Thecell-laden fluid was collected by paracentesis into asterile flask.

Cell fractionation. All cell types were washed twicein sterile, chilled 0.25 M sucrose solution and were thendisrupted with a Teflon pestle homogenizer (Tri-RInstrument Co.) by using the method of Cohn andHirsch (4). The progress of cell disintegration was fol-lowed by examining samples of cell homogenatesunder a phase-contrast microscope. Nuclei and largecytoplasmic fragments were centrifuged into a pelletat 3,000 X g for 20 min at 4 C (nuclear fraction).After the nuclear fraction was removed, lysosomesand some other subcellular particles were collected asa pellet by centrifuging at 15,000 X g for 10 min at4 C (lysosomal fraction). The remaining supernatantfluid was also collected and retained. Samples werefrozen and stored at -20 C.The protein content of cellular fractions was meas-

ured by using the method of Lowry et al. (16).Acid phosphatase assay. An assay for acid phospha-

tase activity was carried out on 0.5 ml of cell fractionto which was added 0.2 ml of 0.125 M ,B-glycerophos-phate and 0.3 ml of 0.3 M acetate buffer (pH 5.0).The reaction was stopped after 15 min at 37 C byadding 0.2 ml of 25%o trichloroacetic acid. The in-organic phosphorus released was measured by themethod of Chen et al. (1), modified so that 1 ml ofassay solution was added to the enzyme-substratemixture. After an additional 2 hr at 37 C, the tubes

815VOL. 5, 1972

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

OUTI7ERIDGE, OSEBOLD, AND ZEE

were centrifuged to remove the protein precipitate, andthe supernatant fraction was transferred to cuvettesand read in a spectrophotometer at a wavelengthof 660 nm. The value obtained was then adjusted ac-cording to the total volume of the fraction and wasalso related to the acid phosphatase content of 101leukocytes.

Antibody response in immunized sheep. The synthe-sis of immunoglobulin G (IgG) antibodies to L.moniocytogenies was used as an index of antigenicstimulation in the sheep which were the source ofimmune macrophages. A somatic agglutination test formercaptoethanol-resistant antibodies was used ac-cording to the technique of Osebold and Aalund (24).

Antibacterial assay against L. monocytogenes. Agrowth inhibition assay was used similar to thatdescribed by Muschel and Treffers (20). Twofolddilutions were made of the cell fractions in 1 ml ofsterile Zobell's solution (pH 4.6). Listeria were grownin Trypticase soy broth on a shaker at 37 C for 18 hr.A 11o dilution of this culture was then made in freshbroth and incubated until a bacterial optical density(OD) of 0.15 + 0.01 at 640 nm was reached. Thebacteria, which were assumed to be in the log phase,were washed twice in Zobell's solution (pH 7.0), and0.3 ml of this bacterial suspension was added to thedilutions of cell fractions followed by an additional0.7 ml of Zobell's solution (pH 4.6). The pH of thetest mixture in some samples was as high as 5.5 in thefirst tube. After the first few dilutions of the cell frac-tions, the acidity increased to pH 4.6. After incubationfor 1 hr at 37 C, 5 ml of Trypticase soy broth wasadded to each tube. The initial OD of the tubes wasmeasured at 640 nm and the tubes were returned tothe water bath. When the OD in control tubes (bac-teria alone) reached 0.4 to 0.45, the OD of all tubeswas again read. A measure of bacterial growth wasobtained by subtracting initial OD from the final OD.The antibacterial titer was the highest dilution of thefraction at which bacterial growth was 50%X/O or lessthan that of the controls.

Fixation and embedding of cells for electron micros-copy. Cells were fixed in gluteraldehyde and postfixed

with osmium tetroxide (30). They were then stainedwith uranyl acetate and embedded in Maraglas 665(Marblette Corp., Long Island City, N.Y.) for sec-tioning. Sections were examined with an A.E.I. EM 6Belectron microscope.

RESULTS

Distribution of acid phosphatase among thesubcellular fractions of sheep and rabbit neutro-phils and macrophages. A comparative study ofthe effects of leukocyte disruption was carriedout with neutrophils and macrophages fromboth sheep and rabbits. Data from the work ofothers on rabbit cells was also considered inreference to the results obtained here with sheepleukocytes. Because of the close association be-tween acid phosphatase and lysosomes, theamounts of the enzyme activity among the sub-cellular components were used as an index oflysosomal content distribution (7).

Cell counts for each type of leukocyte are

shown in Table 1. Cell preparations were identi-fied according to the major cell type. The amountof acid phosphatase activity was considerablygreater in the sheep macrophages than in the othercells. Table 2 indicates that the enzyme contentwas significantly greater in sheep macrophages at<5%- level of probability when compared to theother cell types.As expected from the findings of other investi-

gators, the per cent of acid phosphatase activityassociated with the lysosomes of rabbit neutro-phils was relatively high (56.4%"). More of theenzyme activity remained with the lysosomes fromrabbit cells than remained with the sheep lyso-somes (Table 2). Only 20.6%/o of the activity re-mained in this fraction of the sheep macrophages.The results suggested that there was leakage ofcontents from lysosomes after mechanical disrup-

TABLE 1. Counlts ofmacerophages anzd neutroplhils obtainedjrom peritonleal cavities oJrabbits ad mnammaryglanzds of sheep

Percentage of cells in differential countsPredominant AMean cell

cell type countNeutrophils Macrophages Lymphocytes Eosinophils

Sheep mammarymacrophages.. .. 2.256 X Ioa ±- 1.712 1.05 it0.68 75.17 ±4 3.39 21.45 ±=1.17 2.33 4t1.27

Rabbit peritonealmacrophages .... 1.90 X 108 -- 0.79 10.97 4- 0.32 82.42 ±- 4.14 6.61 4= 1.41 0.0

Sheep mammaryneutrophils ....... 1.456 X 109 it 0.296 98.36 it0.21 0.90 =+ 0.28 0.48 ==0.16,0.26b

Rabbit peritonealneutrophils ...... 1.14 X 109 -+ 0.58 84.29 ==13.31 14.32 ==12.82 1.39 4=0.20!0.0

a Cell counts and their standard errors calculated as the mean of cell collections from six animals.b Standard error not calculated.

816 INFECT. IMMUNITY

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PHAGOCYTE FRACTIONS AGAINST LISTERIA

tion of the cells. The values for standard errorsdenote that the variation in acid phosphatase ac-

tivity was less among sheep cells from which therelease of acid phosphatase was more consistentlyencountered. The finding that a major portion ofthe lysosome contents was released influenced thedesign of subsequent experiments. Attempts to de-tect antibacterial activity in the leukocyte frac-tions required the testing of both lysosomes andthe lysosome-free supernatant fluid.The release of acid phosphatase by the lyso-

somes appeared to parallel the release of anti-bacterial activity by the lysosomes of rabbit andsheep neutrophils. Preliminary experimentsshowed that both the fractions containing thelysosomes and the post-15,000 X g supernatantfluid contained antibacterial activity toward L.monocytogenes and E. coli. This antibacterialactivity was distributed between the lysosomesand the supernatant fluid in approximately thesame proportion as the acid phosphatase activity.

Antibacterial activity of leukocyte fractions

TABLE 2. Acid phosphatase activity among cellular fractions from sheep and rabbit macrophagesand neutrophils

Mean acid Probability that Percentage of activity amongPredominant phosphatase acid phosphatase cellular fractions

cell type activity per macrophagesexceeds other cells Nuclei Supernatant fluid Lysosomes

Sheep mammarymacrophages. . . 1, 314.9a 6.94 - 3.53b 72.43 4- 7.26 20.63 4- 10.66

Rabbit peritonealmacrophages ...... 287.6 0.05 6.20 4- 3.96 52.40 4- 15.21 41.40 4± 16.54

Sheep mammaryneutrophils ....... 63.2 <0.05 0.88c 76.72 == 11.90 22.50 4± 12.36

Rabbit peritonealneutrophils ....... 155.2 <0.05 2.16c 41.40 d= 17.00 56.44 ±- 18.10

a Nanomoles of inorganic phosphorus released.b Standard errors calculated as the mean of determinations from six animals.c Standard error not calculated

84

Z F24LU

z-z

L -

_ 0

z I

O- ZvU

NO. OF SERA USED TO DETERMINE POINT FOR IMMUNIZED SHEEP8 9 10 3 2 6 5 2 3 3 3 3 2

0 10 20 30 40 50 60 70 80

TIME IN DAYS

FIG. 1. Antigenic stimulation of sheep by Listeria monocytogenes as indicated by the synthesis of mercapto-

ethanol-resistant agglutinating antibodies. Titers are expressed as the reciprocal of the serum dilution. Closedcircles = immunized sheep. Open circles = nonimmunized sheep.

I IIII I I I I I I I

30

DO00

50

st.

25 2nd. 3rd.1mm. 1mm.25

_, I . 1

817'VOL. 5, 1972

4(

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

OUTrERIDGE, OSEBOLD, AND ZEE

TABLE 3. Inihibitory titers for Listeria monocytogenes by nieutrophils obtained from mammary glandsof sheep

Day

-12a

8

23

36

56

Immunized

Nuclei

32b3. 83d

19414.25

2565.58

1287.51

496.87

Supernatantfluid

791.65

247.41

64.2323.31

14.64

Lysosomes

81.85

642.22

200.84

20.38

00.79

Nonimmunized

Nuclei

79c8.13

19416.87

2569.56

g918.52

12814.60

Supernatantfluid

972.74

123.87

325.70

03.76

19.10

Lysosomes

240.76

161.43

161.22

00.91

02.23

a Days from the first immunizing inoculation of L. monocytogenes. Negative value equals days beforeinoculation.

b Geometric mean antibacterial titer per milliliter of undiluted cell fraction obtained from threeimmunized ewes. Titers expressed as the reciprocal of fraction dilutions.

, Geometric mean antibacterial titer per milliliter of undiluted cell fraction obtained from threenonimmunized ewes. Titers expressed as the reciprocal of fraction dilutions.

d Numbers in italic equal the mean protein concentration in milligrams per milliliter of undilutedcell fraction.

from sheep immunized with L. monocytogenes.The cellular immune state was induced by thesubcutaneous inoculation of live virulent L.monocytogenes. Synthesis of mercaptoethanol-resistant (IgG) antibodies was used as an indexof antigenic stimulation (24). Figure 1 shows theserum agglutinating antibody response in sheepreceiving three inoculations of the organism. Theresponse curve is in contrast to the absence ofIgG agglutinating activity plotted for three non-immunized sheep. Previous studies had shown thatthe peritoneal exudate cells of sheep immunizedin this way demonstrated the phenomenon ofcellular immunity in vitro (21).Mammary neutrophils. Mammary neutrophils

were collected at weekly intervals for a period of7 weeks from six sheep. Neutrophils constitutednearly 90% of the cells. Three animals receivedimmunizing inoculations during this period, andthree remained uninoculated.The results of assays for antibacterial activity

from the neutrophil fractions are shown in Table3. As expected, there was anti-Listeria activity inall three cellular fractions. The activity wasgreatest in the nuclear fractions. There was a re-duction of antibacterial activity from the lyso-somes and supernatant fluid of neutrophils col-lected late in the experiment after the animals hadserved repeatedly as cell donors. Conceivablythis might have been related to the lipopolysac-charide used to induce the neutrophils. Wieneret al. (34) found that lipopolysaccharide did not

produce degranulation of neutrophils in vitro,but it did cause some loss of acid phosphatasefrom mouse peritoneal leukocytes if it was ad-ministered to the intact animal. The progressiveloss of antibacterial activity in the sheep mam-mary neutrophils may have been due to increas-ing levels of locally produced antibody to thelipopolysaccharide resulting in phagocytosedantigen-antibody complexes which could causeneutrophil degranulation (27).Mammary macrophages. Mammary macro-

phages were also obtained from the sheep atweekly intervals. The samples contained nearly80% macrophages and less than 4% neutrophils.Electron micrographs of the lysosomal cellularfraction revealed considerable numbers of lyso-somes as electron-dense bodies. There were manyvesicles of different sizes. Many of the emptyvesicles were thought to be lysosomal membraneswhich remained after the loss of their contents.The heterogeneous nature of the preparations wasalso shown by the presence of mitochondria withtheir recognizable internal cristae and by clumpsof ribosomes.The mean antibacterial titers are recorded in

Table 4. Antibacterial assays showed a small,variable amount of activity in the nuclear frac-tions from both immunized and normal animals.Antibacterial activity was not detected in thelysosomal fractions from either group. Thus,there was no indication that the immunizing pro-

818 INFECT. IMMUNITY

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PHAGOCYTE FRACTIONS AGAINST LISTERIA 819

TABLE 4. Inihibitory liters for Listeria moniocytogenies by macrophages obtained from the mammary glantdsof sheep

Day

Immunized

Nuclei

0.55d13 0

0.7228 3

0.6142

62

0

0

0.65

1.25

Supernatantfluid

0

0.141

0

0

0

1.36

0.30

1.19

1.72

Lysosomes

0

0.090

0

0

0

0.28

0.04

0.21

0.21

Nonimmunized

Nuclei

5c

1.080

1.3732

1.530

1.780

1.45

Supernatantfluid

0

0.780

1.320

0.910

2.460

2.28

Lysosomes

0

0.150

0

0

0

0.17

0.09

0.35

0.12

a Days from the first immunizing inoculation of L. montocytogenies. Negative value equals days beforeinoculation.

b Geometric mean antibacterial titer per milliliter of undiluted cell fraction obtained from threeimmunized ewes. Titers expressed as the reciprocal of fraction dilutions.

c Geometric mean antibacterial titer per milliliter of undiluted cell fraction obtained from threenonimmunized ewes. Titers expressed as the reciprocal of fraction dilutions.

d Numbers in italic equals the mean protein concentration in milligrams per milliliter of undilutedcell fraction.

cedure had altered the antibacterial capacity ofthe mammary macrophages.

Peritoneal macrophages. Peritoneal macro-phages from nine immunized sheep were tested.The samples contained approximately 60%macrophages, 14% lymphocytes, and 23%neutrophils. A peritoneal macrophage obtained5 days after intraperitoneal injection of oil isshown in Fig. 2. A feature of the peritonealmacrophages was the extensive development ofthe golgi area and the convolutions and projec-tions of the cytoplasmic membrane, suggestingactivation of the cells. There was a relative paucityof recognizable lysosomes in many of these macro-phages.The antibacterial titers from each cellular frac-

tion are shown in Table 5. There was a smallamount of anti-Listeria activity associated withthe nuclear fractions from three of the animals.No antibacterial activity was detected in thelysosomal and supernatant fractions.Lung alveolar macrophages. Lung alveolar

macrophages were obtained from nine immunizedsheep. The samples contained 93 %- macrophages,5%O lymphocytes, and 1 neutrophils. The ultra-structure of lung alveolar macrophages is shownin Fig. 3. Considerable numbers of lysosomesand other electron-dense bodies were seen. Itwas observed, however, that many of the lungmacrophages were in various stages of degenera-tion. This was in contrast to the structural in-tegrity of macrophages obtained from the peri-toneal cavity and the mammary gland.

Antibacterial assays with lung macrophagesyielded unexpected results. Four of the animalshad high antibacterial titers in all subcellularfractions (Table 5). Peritoneal macrophagesfrom the same animals were inactive or showedonly minimal activity in the nuclear fractions.Mammary macrophages had been tested fromthree of the four positive sheep, and those cellshad also been nearly devoid of antibacterial ac-

tivity. Lung macrophage samples from the fiveremaining sheep demonstrated no antibacterialactivity. Some samples from these nonreactinganimals were tested, both at pH 4.6 and at pH 7,but no activity could be demonstrated. In addi-tion, tests were performed with lung alveolarmacrophages from seven nonimmunized sheep.One of the specimens had a high level of activityin the lysosomal fraction (titer of 512). Thatspecimen and three others had antibacterial ac-tivity in both the nuclear and supernatant fluidfractions.The antibacterial fractions from immunized

sheep number 8 were used in an assay to test fortheir effect on Listeria colony formation in an

agar medium. Colonies formed in numbers com-parable to the controls, indicating that themechanism was not bactericidal, although thelung macrophage fractions inhibited Listeriareplication in broth medium.

Peritoneal exudate cells incubated with avirulentListeria organisms prior to fractionation. Livingavirulent Listeria cells (antigen) were allowed tointeract with living host cells for the purposes of

VOL. 5, 1972

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

OUTrERIDGE, OSEBOLD, AND ZEE

FIG. 2. Peritoneal macrophage of sheep showinglysosomes (Ly), a well developed golgi area (G), andconvolutions of the cytoplasmic membrane. Nucleus(N). X 7,000.

stimulating the release of molecular mediatorsfrom lymphoid cells and to promote the formationof secondary lysosomes in macrophages. Sheep7 and 8 were the source of peritoneal exudatecells (2 X 108 cells) which were incubated withan avirulent isolate of L. monocytogenes (25) inthe presence of fresh autologous serum. Theleukocytes and bacteria were mixed in a ratio of40 bacteria to 1 peritoneal cell, which yielded aphagocytic index of 6 bacteria per phagocyte. Themixtures were incubated in rubber-stopperedtubes at 37 C for 4 hr in a slowly revolving drum.The appearance of the parasite-host cell interac-tion is shown in Fig. 4. Phagosomal membranessurrounded groups of ingested bacteria in whichthere were varying stages in the loss of internalbacterial structure. Active phagocytosis attested

to the viability of the cells and the adequacy ofthe techniques for cell collection and handling.Electron-dense granular material was adjacent tothe Listeria cells. Similar material was seen byLeake et al. (14) in rabbit alveolar macrophagesafter the ingestion of L. monocytogenes.

After incubation, the leukocytes were sedi-mented at 75 X g, washed in Zobell's solution atpH 7, homogenized with a Teflon pestle, and frac-tionated as described previously.The avirulent isolate replicated so slowly that

its presence in the antibacterial assays did notinterfere with the interpretation of the growth ofthe virulent assay strain. Two experiments werecarried out, but no antibacterial activity could bedemonstrated in any of the cell fractions.

DISCUSSIONThe nature of the microbicidal system of

macrophages has not been revealed. Variousstudies on cellular immunity have demonstratedthe enhanced antimicrobial activity of livingmacrophages and the apparent interactions be-tween macrophages and lymphocytes (33). Ulti-mately, however, investigators of cellular im-munity wish to explain the phenomenon on amolecular basis. This investigation was an at-tempt to get beyond studies with living cells andsearch for inhibitory or bactericidal substances ofthe lysosomes of immune macrophages. Macro-phages from immunized animals were used onthe assumption that they might contain more in-hibitory factors than normal macrophages or thatthey would contain additional factors. It wastheorized that active substances of the lysosomesmight bind with cellular components in whole-cellhomogenates, thus rendering them inactive for anantibacterial assay. Therefore, the subcellulargranules were separated from the remainder of thecell components, and their contents were then re-leased by freezing and thawing. Assays were per-formed at acid pH to mimic the environment ofthe phagocytic vacuole. A pH of 4.6 was suitablesince it did not affect the viability of L. mono-cytogenes, whereas further increases in aciditywere inhibitory to the bacterium.The search was not for increased amounts of

acid hydrolases, but for a functional inactivatingsystem that might best be detected in the macro-phages of an antigenically stimulated host whentested against the homologous microorganism.It seemed likely that an inactivating system inmacrophages would arise in consort with molecu-lar mediators released in vivo by specifically sensi-tized lymphoid cells. Lymphocytes in considerablenumbers were in association with the macro-phages collected from the mammary gland (16%)and the peritoneal cavity (15 %). However,

820 INFECT. IMMUNITY

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PHAGOCYTE FRACTIONS AGAINST LISTERIA

TABLE 5. Inihibitioni titers for Listeria moniocytogenies by peritonzeal macrophages anid lhung alveolarmacrophages obtainted from immuniZed sheep

Peritoneal macrophages

Supernatantfluid

01.36

016.19

02.98

020.00

0I.80

01.77

012.90

08.75

010.25

Lysosomes

0

0.820

2.90

0

0

0

0

0

0

0

1.00

5.62

0.55

0.30

3.00

10.95

2.90

Lung alveolar macrophages

Nuclei

5121.54

25612.500

1.040

1.950

20488.35

81921.610

7.750

4.80

Supernatant Lysomesfluid Lyoe

0

0.68644.770

0.800

4.620

204814.00

643.860

5.880

1.33

a Number of days between last immunizing inoculation and macrophage collection.bAntibacterial titer expressed as the reciprocal of fraction dilution.c Numbers in italic equal the protein concentration in milligrams per milliliter of

fraction.d Not done.

1281.05

2561.800

0.470

0.350

512I.64

20480.7750

1.590

0.66

undiluted cell

Listeria antigen was not added to the suspensionsuntil after the cells were disrupted. This couldhave blocked the role of the lymphoid cells.Nonetheless, live macrophages, free of lympho-cytes, have been shown to function as "immune"cells in vitro. In the technique of Mackaness (18),explants of peritoneal cells from immunized micewere washed free of lymphocytes, cultivated invitro for 24 hr, and then exposed to L. mono-

cytogenes. It was reported that virtually every cellin monolayers prepared from such mice wascapable of inactivating ingested Listeria. Thiswas a circumstance that would require transferof a chemical mediator at a time prior to intimateinteraction with the bacterium in the assay. Con-sequently, it seemed likely in our studies that themacrophages could have either stored or synthe-sized antibacterial components when collected asearly as 5 days after the sheep were stimulatedwith living L. monocytogenes.The lysosomal fractions of immune macro-

phages from three tissue sites were found to begenerally inactive in assays for growth inhibitionof L. monocytogenes. Antibacterial activity wasencountered in some nuclear fractions and in thelysosomal fractions of sheep and rabbit neutro-

phils. These results were thought to be due tohistones in the nuclei and basic proteins in thelysosomes, respectively. Zeya and Spitznagel (38)have associated antibacterial activity with thebasic proteins in the specific granules of neutro-phils, but similar proteins could not be demon-strated in the lysosomes of rabbit macrophages(12). Thus, the factors responsible for intracellu-lar killing in macrophages were not revealed bythe isolation of the subcellular particles of thecells and release of their contents.The presence ofa factor inhibitory to the growth

of Listeria in the cellular fractions from some ofthe sheep lung macrophages was not in accordwith the general pattern of experimental results.The possibility exists that an additional mechanismfor microbial inhibition may be present in lungmacrophages. There was no obvious evidence ofpneumonitis which could be correlated with theactive lung macrophages. The role of nuclearhistones might be considered as the basis for theactivity because of the degenerated appearance ofmany lung macrophages observed by electronmicroscopy. But this seemed unlikely in view ofthe results with the peritoneal macrophages andseveral of the other lung cell preparations. It was

Sheepno.

2

3

4

5

6

7

8

9

10

Days'

24

8

11

7

13

5

6

5

8

12

-Nuclei

16'2. 77c

430.00_d

03.99

036.75

06.56

02.44

020.62

025.20

821.50

821VOL. 5, 1972

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

OUTTERIDGE, OSEBOLD, AND ZEE

FIG. 3. Ltiung alveolar niacrophlage of slheep conttaintintg sevecral lysosomes (Ly). Ni nwerous mitochondria (M)anid ribosornes are also seent. Nuicleus (N). X 23,000.

reported by Holub and Hauser (11) that somelung alveolar histiocytes in the rabbit produceantibody. However, by immunoelectrophoreticexamination we were unable to detect immuno-globulin of any class in the three cell fractions oflung macrophages. Perhaps the substance wehave demonstrated in some of the lung macro-phage homogenates is comparable to "myco-

suppressin" found by Youmans in homogenatesof the lungs of guinea pigs and rabbits im-munized with BCG. Mycosuppressin inhibitedthe respiration and growth of Mycobacterium tu-herculosis. The inhibitor to L. monocytogenes rep-lication found in the alveolar macrophages ofsheep was not bactericidal, and there was no ap-parent correlation of its presence with the immune

822 INFECT IMMrUNITY

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PHAGOCYTE FRACTIONS AGAINST LISTERIA

FIG. 4. Phagocytosed Listeria monocytogenzes inz a peritoneal macrophage of sheep. Electron-dense granumlarmaterial surrounzds the groups of intgested bacteria withinz phagosomal membranies. Nucleuts (N). X 21,000.

status of the host since it was not detected in thecells of all immunized sheep and was also en-countered in one of seven nonimmunized speci-mens.Our finding that not all of the acid phosphatase

activity in rabbit and sheep macrophages is as-sociated with the lysosomes is in conformity withthe work of Leake and Myrvik (15) with rabbitalveolar macrophages. They also used the Teflonpestle method to disrupt the cells and found thatfrom 34.1 to 49.8%o of the total acid phosphataseactivity was associated with the cellular fractioncontaining the lysosomes. These figures agree

with those found for acid phosphatase activityassociated with the granules of rabbit peritonealmacrophages (41.4%, ±16.54) in our experi-ments. In the sheep mammary macrophages,however, only 20.63%, ±10.66, was found to beassociated with the pellet containing the lyso-somes. As a matter of comparative biology it ap-peared that the lysosomes of sheep macrophagesand neutrophils were more easily disrupted thanthose of the rabbit.One set of experiments was designed to en-

hance the opportunity for the participation ofmolecular mediators from lymphocytes. Peri-

823VOL. 5, 1972

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

824 OUTTERIDGE, OSEBOLD, AND ZEE

toneal exudate cells (60%c, macrophages, 14%lymphocytes) from L. monocytogenes-immunizedsheep were allowed to interactwith a live, avirulentmutant of L. monocytogenes. The purpose was toinduce the release of lymphokines and to stimu-late secondary lysosome formation in the macro-phages. Although this milieu of interacting cellu-lar components did not reveal a microbial in-hibitory system, the works of other investigators(9, 28) indicate the role of the "immune lympho-cyte" in cell-mediated immunity to infectiousagents.

Explanations of the mechanisms for microbialinactivation by neutrophils are emerging fromrecent studies. The cationic proteins of the lyso-somes (38) and the myeloperoxidase system ap-pear to be central in importance (29). It seems aperversity that the macrophage has been so dif-ficult to understand. The dissimilarity of anti-microbial mechanisms between the neutrophil andthe macrophage appears to point to the unique-ness of the macrophage and its need to play adifferent biological role. The results of the experi-ments reported here emphasize the complexity ofthe antimicrobial system and the need to searchfor novel interactions between lymphocytes andmacrophages in the state of cellular immunity.

ACKNOWLEDGMENTS

We thank Caroline Kirkham and Renee Seely for their tech-nical assistance.

This investigation was supported by Public Health Servicegrant Al 06895 from the National Institute of Allergy and In-fectious Diseases.

LITERATURE CITED

1. Chen, P. S., T. Y. Toribara, and H. Warner. 1956. Micro-determination of phosphorus. Anal. Chem. 28:1756-1758.

2. Cohn, Z. A. 1963. The fate of bacteria within phagocyticcells. 1. The degradation of isotopically labeled bacteriaby polymorphonuclear leucocytes and macrophages. J.Exp. Med. 117:27-42.

3. Cohn, Z. A., and B. Benson. 1965. The differentiation ofmononuclear phagocytes. Morphology, cytochemistry andbiochemistry. J. Exp. Med. 121:153-170.

4. Cohn, Z. A., and J. G. Hirsch. 1960. The influence of phago-cytosis on the intracellular distribution of granule-associ-ated components of polymllorphonucleair leucocytes. J.Exp. Med. 112:1015-1022.

5. Coppel, S., and G. P. Youmans. 1969. Specificity of theanamnestic response produced by Listeria monocytogenesor Mycobacterium tuberculosis to challenge with Listeriarnoniocytogenies. J. Bacteriol. 97:127-133.

6. Dannenberg, A. M. 1968. Cellular hypersensitivity and cellu-lar immunity in the pathogenesis of tuberculosis: speci-ficity, systemic and local nature, and associated macro-phage enzymes. Bacteriol. Rev. 32:85-102.

7. de Duve, C. 1963. The lysosome concept, p. 1-31. In A. V. S.de Reuck and M. P. Cameron (ed.), Lysosomes. LittleBrown & Co., Boston.

8. Elberg, S. S., P. Schneider, and J. Fong. 1957. Cross-immunitybetween Brucella melitensis and Mycobacterium tuberculosis.Intracellular behaviour of Brucella melitensis in monocytesfrom vaccinated animals. J. Exp. Med. 106:545-554.

INFECT. IMMUNITY

9. Godal, T., R. J.W . Rees, and J. 0. Lamvik. 1971. Lym pho-cyte-mediated modification of blood-derived macrophagefunction in vitro; inihibition of growth of intracellularMycobacteria with lymphokines. Clin. Exp. Immunol.8:625-637.

10. Heise, E. R., Q. N. Myrvik, and E. S. Leake. 1965. Effect ofBacillus Calmette-Guerin on the levels of acid phospha-tase, lysozyme and cathepsin in rabbit alveolar macro-phages. J. Immunol. 95:125-130.

11. Holub, M., and R.E. Hauser. 1969. Lung alveolar histio-cytes engaged in antibody production. Immunology 17:207-226.

12. Janoff, A., M. A. Bean, andE. Schuller. 1965. Mediators ofinflammation in leukocyte lysosomes.Ill. Studies on lyso-somes of rabbit peritoneal macrophages. Life Sci. 4:2361-2372.

13. Kurashige, S., N. Osawa, M. Kawakami, and S. Mitsuhashi.1967. Experimental salmonellosis. X. Cellular immtunityand its antibody in mouse mononuclear phagocytes. J.Bacteriol. 94:902-906.

14. Leake, E. S., D. G. Evans and Q. N. Myrvik. 1971. Ultrat-structural patterns of bacterial breakdown in normal andgranulomatous rabbit alveolar macrophages. Res. J.Reticuloendothel. Soc. 9:174-199.

15. Leake, E. S., and Q. N. Myrvik. 1964. Differential release oflysozyme and acid phosphatase from sub-cellular granulesof normal rabbit alveolar macrophages. Brit. J. Exp.Pathol. 45:384-392.

16. Lowry, 0. H.. N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

17. Lurie, M. B. 1942. Studies on the mechanism of immunity intuberculosis. The fate of tubercle bacilli ingested bymono-nuclear phagocytes derived from normal and immunizedanimals. J. Exp. Med. 75:247-268.

18. Mackaness, G. B. 1962. Cellular resistance to infection. J.Exp. Med. 116:381-406.

19. Mackaness, G. B. 1968. The immunology of antituberculousimmunity. Amer. Rev. Resp. Dis. 97:337-344.

20. Muschel, L. H., and H. P. Treffers. 1956. Quantitative studieson the bactericidal actions of serum and complement. I.A rapid photometric growth assay for bactericidal activity.J. Immunol. 76:1-10.

21. Njoku-Obi, A. N., and J. W. Osebold. 1962. Studies onmechanisms of immunity in listeriosis. I. Interaction ofperitoneal exudate cells from sheep with Listeria m71on1o-cytogentes in vitro. J. Immunol. 89:187-194.

22. North, R. J. 1966. The localization by electron microscopyof acid phosphatase activity in guinea pig macrophages.J. Ultrastruct. Res. 16:96-108.

23. North, R. J., and G. B. Mackaness. 1963. Electron micro-scopical observations on the peritoneal macrophages ofnormal mice and mice immunized with Likteria monocYto-gentes. I. Structure of normal macrophages and the earlycytoplasmic response to the presence of ingested bacteria.Brit. J. Exp. Pathol. 44:601-607.

24. Osebold, J. W., and 0. Aalund. 1968. Interpretationi ofserum agglutinating antibodies to Listeria monzocytogentesby immunoglobulin differentiation. J. Infec. Dis. 118:139-148.

25. Osebold, J. W., P. M. Outteridge, and L. D. Pearson. 1970.Mutation of Listeria monzocytogenzes after prolonged invivo survival in diffusion chambers. Infect. Immunity1:209-211.

26. Outteridge, P. M., J. W. Osebold, and Y. C. Zee. 1971.The mammary gland of the ewe as a source of neutrophilsand macrophages for repeated collection. Res. J. Reticulo-endothel. Soc. 10:440-448.

27. Outteridge, P. M., J. D. Rock, and A. K. Lascelles. 1965.The immune response of the mammary gland and regionallymph node following antigenic stimulation. Aust. J. Exp.Biol. Med. Sci. 43:265-274.

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

PHAGOCYTE FRACTIONS AGAINST LISTERIA

28. Patterson, R. J.. and G P. Youmans. 1970. Demonstrationin tissLe c:.lture of lymphocyte-mediated immuniity totubsrculosis. Infect. Immunity 1:600-603.

29. Paul. B. B.. R. R. Strauss, A. A. Jacobs, and A. J. Sbarra.1970. Function of H202, myeloperoxidase, and hexosemonophosphate shunt enzymes in phagocytizing cellsfrom different species. Infect Immunity 1:338-344.

30. Robbins, E., and N. K. Gonatas. 1964. In vi.ro selection ofthe mitotic cell for subsequent electron microscopy. J.Cell Biol. 20:356-359.

31. Rowley, D. 1966. The carrier state and cellular immunity.Experientia 22:9-13.

32. Saito, K., T. Akiyama, N. Nakano, and D. Ushiba. 1960.Interaction between Salmontella enteritidis and tissuecultured imiacrophages derived from immunized animals.

Jap. J. Microbiol. 4:395-407.33. Weiser, R. S. 1971. Antitissue versus antimicrobial cellular

immunity: a perspective. Res. J. Reticuloendothel. Soc.10:17-27.

34. Wiener, E., A. Beck, and M. Shilo. 1965. Effect of bacterial

lipopolysaccharides on mouse peritoneal leukocytes. Lab.Invest. 14:475-487.

35. Youmans, A. S.. and G. P. Youmans. 1962. Effect of myco-

suppressin on the respiration and growth of Mycobacteriumtuberculosis. J. Bacteriol. 84:708-715.

36. Youmans, G. P. 1971. The role of lymphocytes and otherfactors in antimicrobial cellular immunity. Res. J. Reticulo-endothel. Soc. 10:100-1 19.

37. Zeya, H. I., and J. K. Spitznagel. 1966. Cationic proteins ofpolymorphonuclear leukocyte lysosomes. I. Resolution ofantibacterial and enzymatic activities. J. Bacteriol. 91:750-754.

38. Zeya, H. I., and J. K. Spitznagel. 1969. Cationic protein-bearing granules of polymorphonuclear leukocytes: sepa-

ration from enzyme-rich granules. Science 163:1069-1071.39. Zobell. C. E., and M. H. Zobell. 1932. Metabolism studies

on the Brucella group. III. Viability in aqueous solutions.J. Infect. Dis. 50:538-541.

40. Zucker-Frankliji, D., and J. G. Hirsch. 1964. Electron micro-scope studies on the degranulation of rabbit peritonealleukocytes during phagocytosis. J. Exp. Med. 120:569-576.

VOL. 5, 1972 825

on Novem

ber 3, 2020 by guesthttp://iai.asm

.org/D

ownloaded from